Discovery and Characterization of Novel 2-Phosphoglycerate Kinase and Cyclic 2,3-Diphosphoglycerate Synthase from Thermophilic (Meta)Genomes

2-Phosphoglycerate kinase (2PGK) and cyclic 2,3-diphosphoglycerate synthase (cDPGS) are key enzymes involved in the biosynthesis of cyclic 2,3-diphosphoglycerate (cDPG), an extremolyte known to stabilize proteins in hyperthermophilic Archaea. Using bioinfor- matics approaches, two candidate genes for each enzyme were identifi ed from a range of thermophilic bacterial and archaeal genomes and metagenomes. Signifi cantly, one gene pair derived from the Taman mud volcano metagenome represents the fi rst indication of a bacterial cDPG biosynthesis pathway. The recombinant expression and purifi cation of these enzymes paved the way to their biochemical and structural characterization. One 2PGK candidate displayed predominant ATPase activity, while the newly identifi ed cDPGS variants demonstrated cDPG synthase activity. Moreover, one of the latter biocatalysts, Ts- cDPGS from the hyperthermophilic archaeon Thermococcus sibiricus, demonstrated a notable thermostability and its 3D structure was resolved at a resolution of 2.2 Å. These fi ndings broaden our understanding of extremophilic enzyme systems and lay the foundation for biotechnological applications involving extremolyte production.